Patients suffering from failure of a vital organ can be treated with whole organ transplantation; transplantation alone, however, cannot meet the public's medical needs due to the limited supply of donated organs. Artificial tissues present an alternative to donated organs, but our inability to engineer functional microvessels within such constructs broadly prevents the development of clinically effective artificial tissues. Because all tissue- scale regenerative therapies require perfusion, the ability to form functional microvasculature is paramount. Upon implantation of bulk artificial tissues without microvasculature, cells on the inside die from lack of oxygen, leaving a shell of live cells about 0.2 mm thick. Artificial vascularization will prevent this by creating a volume- spanning perfusion-competent network and by enabling swift vascular integration after implantation. To engineer microvasculature, we propose a novel biomaterials strategy: nanoscale clustering of cell- matrix adhesion ligands. Previous work on 2D surfaces has shown that clustering of ligands increases growth factor sensitivity and motility via receptor clustering. Research using animal models has shown that expression of molecular disruptors of receptor clustering is associated with a decrease in both branching and maturation. Though ligand clustering and receptor clustering are related thermodynamically, it is unknown whether nanoscale ligand clustering will lead to morphologically appropriate microvasculature in a 3D, bulk biomaterial. To answer this question, we have developed a nanofibrous biomaterial that can be fabricated at a specified bulk concentration and nanoscale clustering of adhesion ligands. By mimicking the nanoscale order of the native the extracellular matrix, we expect to achieve organotypic blood-vessel structure formation in vitro. We specifically hypothesize that clustering of adhesion ligands will upregulate three essential cellular process that lead to formation of blood-vessels in vivo: (1) growth factor sensitivity, (2) cell motility, and (3) vessel branching and maturation. Growth factor sensitivity will be assessed by measuring proliferation, metabolic activity, and protease secretion. Motility, as parameterized by cell speed and persistence length, and cytoskeletal organization will be assessed by quantitative image analysis. Branching and maturation will be assessed by immunostaining for appropriate markers and computational analysis of morphological data. The biomaterials proposed here can be further developed as an implant for regenerative medicine by incorporating non- overlapping technologies such as co-culture of tissue-specific stems cells, growth factor delivery, and bioreactor/ mechanical stimulation. My mentor Sarah Heilshorn, an expert in protein-based materials engineering, and our collaborator John Cooke, a senior professor of microvascular signaling biology, have developed an appropriate training plan to accomplish this project. PUBLIC HEALTH RELEVANCE: There are 112,000 Americans waiting for donated organs, and, in 2008, 9% of those on the waitlist died before a transplant became available. As an alternative to donated organs, artificial tissue has yet to become clinically useful because we cannot meet the perfusion needs of bulk constructs post-implantation. Induction of blood-vessels within artificial tissues, which we propose to achieve using a novel biomaterials strategy, will overcome this deficiency by forming a perfusion-ready network and by enabling swift vascular integration upon implantation.